A single isolated smooth muscle fiber preparation will be employed to investigate the organization and functtonal properties of the contractile apparatus, the ionic basis of electrical activity, the action of neurotransmitters and the role of cyclic nucleotides in regulating contraction. Large smooth muscle cells will be isolated enzymatically from the stomach muscularis of Bufo marinus. Single smooth muscle cells will be mounted for isometric measurement of force. The basis for the length-dependence of force generation will be probed using correlated measurements of ultrastruture, birefringence, force, and dynamic stiffness. "Cross-bridge" properties will be assessed from mechanical transients resulting from rapid length changes. The event(s) responsible for the long latencies for force development will be investigated utilizing birefrigence, temperature sensitivity and the effect of intracellular microiontophoresis of various cations to pinpoint the cause for the unusually long delay in smooth muscle. The electrical properties of the cell membrane and the ionic basis of the resting potential and action potential will be studied with intrcellular recording and current injection in a single isolated cell. The action of neurotransmitters and relaed substancies and the disposition of receptor on the surface of a single cell wil be studied using microionotophoretic techniques. The relationship between electrical and mechanical activity will be examined in a single cell. Purified cell suspensions will be used for measures of ionic flux and ion concentration in order to determine the mechanisms responsible for the normal distribution of ions and the ionic events underlying the response to neurotransmitter-like substances. The suspensions will also be used for biochemical assays of the level of cyclic nucleotides whose role in regulating contractile activity will be investigated. The part played by specific protein phosphorylation in mediating the responses to catecholamines will be studied with suspensions of cell "ghosts" and membrane preparations.